Ultra-sensitive isotope probing to quantify activity and substrate assimilation in microbiomes

Abstract

Background Stable isotope probing (SIP) approaches are a critical tool in microbiome research to determine associations between species and substrates, as well as the activity of species. The application of these approaches ranges from studying microbial communities important for global biogeochemical cycling to host-microbiota interactions in the intestinal tract. Current SIP approaches, such as DNA-SIP or nanoSIMS allow to analyze incorporation of stable isotopes with high coverage of taxa in a community and at the single cell level, respectively, however they are limited in terms of sensitivity, resolution or throughput. Results Here, we present an ultra-sensitive, high-throughput protein-based stable isotope probing approach (Protein-SIP), which cuts cost for labeled substrates by 50–99% as compared to other SIP and Protein-SIP approaches and thus enables isotope labeling experiments on much larger scales and with higher replication. The approach allows for the determination of isotope incorporation into microbiome members with species level resolution using standard metaproteomics liquid chromatography-tandem mass spectrometry (LC–MS/MS) measurements. At the core of the approach are new algorithms to analyze the data, which have been implemented in an open-source software (https://sourceforge.net/projects/calis-p/). We demonstrate sensitivity, precision and accuracy using bacterial cultures and mock communities with different labeling schemes. Furthermore, we benchmark our approach against two existing Protein-SIP approaches and show that in the low labeling range used our approach is the most sensitive and accurate. Finally, we measure translational activity using 18O heavy water labeling in a 63-species community derived from human fecal samples grown on media simulating two different diets. Activity could be quantified on average for 27 species per sample, with 9 species showing significantly higher activity on a high protein diet, as compared to a high fiber diet. Surprisingly, among the species with increased activity on high protein were several Bacteroides species known as fiber consumers. Apparently, protein supply is a critical consideration when assessing growth of intestinal microbes on fiber, including fiber-based prebiotics. Conclusions We demonstrate that our Protein-SIP approach allows for the ultra-sensitive (0.01 to 10% label) detection of stable isotopes of elements found in proteins, using standard metaproteomics data.

Microbial Growth under Limiting Conditions-Future Perspectives

“…Some methods used to this aim include the use of nalidixic acid (which elongates active bacteria and inhibits cell division) [ 129 ], measurements of membrane potential (to differentiate metabolically active and inactive cells) [ 130 ], FDA hydrolysis (fluorescein diacetate; a fluorogenic compound that fluoresces after enzymatic hydrolysis) [ 131 ], microautoradiography using radiolabeled substrates (glucose, thymine, amino acids, etc.) [ 132 ] or stable isotope labeling as in NanoSIMS [ 82 , 95 ], among multiple other procedures [ 96 , 124 ].…”

“…Methodology is needed in order to determine the growth status and/or growth rate of specific bacterial taxa in complex environmental communities. Isotopic-labeling techniques measuring the incorporation of specific labeled substrates during a relatively short incubation period are among the possibilities for estimating the bacterial activity of specific bacteria and their metabolism [ 94 , 95 ]. Molecular methods targeting DNA and/or RNA are another option.…”

“…At present, only a very few model species or bacterial groups have been studied [ 96 ]. Additional perspectives have been provided using NanoSIMS [ 82 , 95 ], which could add relevant information for single-cell functional studies on natural samples. Methods that provide important information through rapid tests and measurements will be greatly welcomed in the years to come, enabling most of the issues mentioned herein to start being solved.…”

More than the sum of its parts: uncovering emerging effects of microbial interactions in complex communities

“…While on the level of incorporation of nucleic acids, the flow of a labelled substrate across different trophic levels is challenging, protein-SIP allows to quantify the relative isotopic abundances in an organism's proteome and thus to draw conclusions about trophic interactions such as predation and cross-feeding between microorganisms. Recently, an ultra-sensitive, accurate and high-throughput protein-SIP method was described that allows for precise detection of translation, measuring activity in microbial communities (Kleiner et al 2023 ). The more sensitive and accurate labeling-based methods become, the more informative they are for studying complex interaction networks in microbial communities.…”

Can we manage microbial systems to enhance carbon storage?

“…However, to do so, we must move beyond correlative studies and develop predictive, experimentally validated and quantitative insights to translate our understanding of ecological phenomena into actionable strategies for managing microbial systems and C turnover (Bell, 2019; Prosser, 2022; Widder et al, 2016). To achieve a mechanistic understanding, we must continue to develop methods and techniques that trace C fluxes through microbial systems, such as stable isotope techniques (Beaupré et al, 2016; Hungate et al, 2015; Kleiner et al, 2023; Wilhelm et al, 2022). Deeper ecological insights can also be acquired with molecular techniques, such as tn‐Seq and ET‐seq (Rubin et al, 2022; van Opijnen et al, 2009), which manipulate microbial systems to understand the function of genes and pathways in C turnover at the community level and in environmental contexts.…”